The cell envelope of E. coli is a complex structure composed of three layers - an inner and outer membrane with a thin layer of peptidoglycan (PG) sandwiched between. Bacteria must carry out the delicate task of expanding, remodeling, and degrading PG to grow and divide, all while maintaining the integrity of this essential stress-bearing structure (1). Any perturbations to the process of cell wall growth can lead to a catastrophic breach in the cell wall and ultimately cause cell lysis. As such, many clinically valuable antibiotics, including penicillin and other ss-lactams, target this unique molecule by inhibiting proteins required for PG biogenesis. Despite the considerable knowledge we have acquired about the cell wall over the past several decades, many aspects of its biogenesis remain to be elucidated. This is highlighted by the fact that approximately one- third of proteins in E. coli are associated with the cell envelope, but half of those proteins have unknown functions (2). To further expand our knowledge of the cell wall and fully explore the potential array of targets for future antibiotics, it is of utmost importance to focus additional studies on he uncharacterized genes connected to the cell envelope. Using a genetic screen for the identification of new cell morphogenesis factors in E. coli, I implicated the gene of unknown function, yceG, as a potential cell wall remodeling factor. My preliminary biochemical experiments show that YceG is indeed a novel cell wall hydrolase. Bioinformatic analysis indicates that yceG is evolutionarily conserved across many other gram-negative and gram-positive bacteria. The proposed experiments aim to 1) determine the enzymatic specificity of YceG in vitro, 2) to define the physiological role of YceG in vivo, and 3) to explore how YceG is regulated. Knowledge of the function and regulation of this unique cell wall hydrolase will provide insight necessary to uncover new vulnerabilities in the cell wall assembly process that can be targeted by much needed novel antibacterial therapies.